Fig. 4. The PH domain is required for phosphorylation of Ser277. (A) Schematic of mutants used for the mapping of domains that are indispensable for AFAP-110 phosphorylation on Ser277. P71, Pro71 of the SH3-interaction domain; 71A, Pro71 to Ala mutation; PH, pleckstrin homology; LZ, leucine zipper; ABD, actin-binding domain. (B) Western blot analysis of AFAP-110 mutants described in Fig. 4A. 48 hours post transfection cells were treated for 1 hour with 100 nM PMA as indicated, directly lysed in sample buffer and SDS-PAGE and western blot analyses were performed. Western blot results using anti-pSer277 antibody ((P) AFAP; upper panel), and anti-GFP antibody (lower panel) to control for equal loading. Molecular mass markers on the left of the blots.